Which hormone interacts with membrane bound receptor and does not normally enter the target?

Membrane Receptors/Channels for Cough Transduction

Extensive studies exist on the sensory nerve membrane channels and receptors involved in coughing.12–14 The details are complex, so they are summarized only briefly. However, the results are of considerable importance as they indicate how chronic coughing manifests and provide a rationale for the antitussive therapeutics available or in development.

Vagalnociceptors express severaltransient receptor potential (TRP) channels. Capsaicin and temperature-sensitivetransient receptor potential vanilloid-1 (TRPV1) receptors are directly activated by capsaicin, used commonly as a tussive agent in clinical studies of cough; these receptors are additionally sensitized or indirectly activated by heat, protons, bradykinin, arachidonic acid derivatives,adenosine triphosphate (ATP), and phosphokinase C. TRPV4 is osmotically sensitive and mostly found on epithelial cells but also on a subset of vagal sensory nerve fibers, whereas TRP ankyrin 1 is often coexpressed with TRPV1 on many vagal nociceptors in the airways and is activated by allyl isothiocyanate (mustard oil), cinnamaldehyde (from cinnamon), and acrolein (from cigarette smoke). C fibers also express tetrodotoxin-insensitive voltage-gated sodium channels, many G protein–coupled receptors for inflammatory mediators, and receptors for neurotrophins, such as nerve growth factor.

Vagalmechanosensors are thought to express mechanically gated membrane ion channels that are unique to this class of airway sensory fibers but have yet to be identified. In addition, they have other channels that can be activated by acids and belong to theacid-sensing ion channel (ASIC) family. They possess different types of voltage-sensitive ion channels needed for action potential formation and conduction, including the NaV1.7 subtype of voltage-gated sodium channels, characterized by sensitivity to the neurotoxin tetrodotoxin. However, the vagal mechanosensors lack several TRP channels found in vagal nociceptors, although these TRP channels may be induced during inflammation.

Both nociceptors and mechanosensors often also express receptors for ATP and other purines. Of note, P2X2 and P2X3 purinergic receptors can be expressed alone or in combination, and it is thought that local release of ATP from the injured or inflamed airways is a major contributor to cough hypersensitivity syndrome acting via these receptors.3

15.10 Highlights

Cell plasma membranes (and a few intracellular membranes as well) contain membrane receptors. These receptors mediate signal transduction for cellular responses to extracellular stimuli. Membrane receptors are usually transmembrane proteins. Transmembrane proteins with part of their mass on both sides of the membrane are poised structurally to transmit information from one side of the membrane to the other.

The domain of the receptor exposed to the external medium often has a binding site for a ligand. Ligands can be hormones, neurotransmitters, lipoproteins, transferrins, extracellular matrix, and a wide variety of other molecules. The domain of the receptor exposed to the cytoplasm has functionality to activate intracellular proteins such as kinases, G proteins, guanylate cyclase, ion transporters, among a myriad of other functionalities. In general ligand binding to the extracellular domain causes conformational changes that are transmitted to the intracellular domain and initiate intracellular changes as a result.

The LDL receptor and transferrin receptors are examples of receptors that function by receptor-mediated endocytosis. The LDL receptor is a transmembrane protein of the plasma membrane. Occupancy of the ligand binding site of the LDL receptor in the extracellular domain of the receptor by LDL initiates endocytosis. Clathrin-coated vesicles transport the receptor–LDL complex to endosomes within the cell. The receptor and LDL are separated and the LDL is ultimately catabolized, which can lead to regulation of cholesterol biosynthesis while the receptor can be recycled to the cell surface.

The insulin receptor is a transmembrane protein in the plasma membrane functioning as a dimer. The insulin receptor modulates the cellular response to insulin, through insulin binding to the extracellular domain of the receptor. Insulin binding stimulates kinase activity that leads, among other results, to the autophosphorylation of the receptor. One of the responses of cells to activation of the insulin receptor is the recruitment of glucose transporters to the plasma membrane leading to increased glucose transport into the cell.

The nicotinic acetylcholine receptor is a transmembrane protein consisting of five subunits. The receptor is a ligand-gated cation channel that responds to the neurotransmitter, acetylcholine, at the synapse between nerve and muscle. Occupancy of the ligand binding site by acetylcholine leads to an opening of the sodium channel (that is part of the receptor) and a depolarization of the plasma membrane.

Integrins are receptors in the plasma membranes of cells. They are multisubunit transmembrane proteins. They have large extracellular domains, consisting of individually structured subdomains covalently linked by flexible linkers. The transmembrane domain is a single α-helix and the intracellular domain is small. Integrins play a major role in cellular attachment to extracellular matrix proteins. Intracellular responses include tyrosine kinase activation and modulation of intracellular calcium.

G-protein coupled receptors are a large class of important cell surface receptors. They have become major drug targets. These receptor systems consist of three major components: the ligand, the transmembrane receptor, and the G protein. G-protein coupled receptors are usually found in the plasma membrane. The receptor binds a ligand from outside the cell. This binding causes a conformational change in the receptor such that the conformation of the cytoplasmic face of the receptor is altered. The receptor can then bind the G protein, a heterotrimer from the inside of the cell. These G proteins become activated after binding to an activated receptor. The subunits of the G proteins disassociate, and the Gα subunit and separately the Gβγ subunit activate target proteins to alter behavior of the cell. Target enzymes include phospholipases, adenylate cyclase, and phosphodiesterases, among others.

Receptors

All signaling molecules bind to specific receptors that act as signal transducers, thereby converting a ligand-receptor binding event into intracellular signals that affect cellular function. Receptors can be divided into four basic classes on the basis of their structure and mechanism of action: (1)ligand-gated ion channels, (2)G protein–coupled receptors (GPCRs), (3)enzyme-linked receptors, and (4)nuclear receptors (Table 3.1;Figs. 3.4 and3.5).

Ligand-gated ion channels mediate direct and rapid synaptic signaling between electrically excitable cells (seeFig. 3.4A). Neurotransmitters bind to receptors and either open or close ion channels, thereby changing the ionic permeability of the plasma membrane and altering the membrane potential. For examples and more details, seeChapter 6.

GPCRs regulate the activity of other proteins, such as enzymes and ion channels (seeFig. 3.4B). In the example inFig. 3.4B, the interaction between the receptor and the target protein is mediated by heterotrimeric G proteins, which are composed of α, β, and γ subunits. Stimulation of G proteins by ligand-bound receptors activates or inhibits downstream target proteins that regulate signaling pathways if the target protein is an enzyme or changes membrane ion permeability if the target protein is an ion channel.

Enzyme-linked receptors either function as enzymes or are associated with and regulate enzymes (seeFig. 3.4C). Most enzyme-linked receptors are protein kinases or are associated with protein kinases, and ligand binding causes the kinases to phosphorylate a specific subset of proteins on specific amino acids, which in turn activates or inhibits protein activity.

Nuclear receptors are small hydrophobic molecules, including steroid hormones, thyroid hormones, retinoids, and vitamin D, that have a long biological half-life (hours to days), diffuse across the plasma membrane, and bind to nuclear receptors or to cytoplasmic receptors that, once bound to their ligand, translocate to the nucleus (seeFig. 3.5). Some nuclear receptors, such as those that bind cortisol and aldosterone, are located in the cytosol and enter the nucleus after binding to hormone, whereas other receptors, including the thyroid hormone receptor, are located in the nucleus. In both cases, inactive receptors are bound to inhibitory proteins, and binding of hormone results in dissociation of the inhibitory complex. Hormone binding causes the receptor to bind coactivator proteins that activate gene transcription. Once activated, the hormone-receptor complex regulates the transcription of specific genes. Activation of specific genes usually occurs in two steps: an early primary response (≈30 minutes), which activates genes that stimulate other genes to produce a delayed (hours to days) secondary response (seeFig. 3.5). Each hormone elicits a specific response that is based on cellular expression of the cognate receptor, as well as on cell type–specific expression of gene regulatory proteins that interact with the activated receptor to regulate the transcription of a specific set of genes (seeChapter 38 for more details). In addition to steroid receptors that regulate gene expression, evidence also suggests the existence of membrane and juxtamembrane steroid receptors that mediate the rapid, nongenomic effects of steroid hormones.

Cell-Surface Receptors Involved in Signal Transduction

Membrane receptors for transmitters, peptides, and pharmacological agents are central to signal transduction. They selectively recognize chemical effectors (neuronal or hormonal) and allosterically transduce binding recognition into biological action though the activation of ligand-gated ion channels (LGICs) and/or G-protein-coupled receptors (GPCRs). Various features of membrane receptors for neurotransmitters are well accommodated by the MWC model. These receptor proteins are typically heterooligomers and exhibit transmembrane polarity. In general, the regulatory site to which the neurotransmitter binds is exposed to the synaptic side of the membrane while the biologically active site is either a transmembrane ion channel, a G-protein-binding site, or a kinase-catalytic site facing the cytoplasm. Interactions between the two classes of sites are mediated by a transmembrane allosteric transition. Signal transduction or activation is mediated by a concerted cooperative transition between a silent resting state and an active state. Agonists stabilize the active state and competitive antagonists the silent state, and partial agonists may bind nonexclusively to both. These receptors can also undergo a cascade of slower, discrete allosteric transitions, which include refractory regulatory states that result in the desensitization or potentiation of the physiological response.

Membrane receptors couple to signaling pathways utilizing diverse mechanisms

Some membrane receptors - for example, the β-adrenergic receptors or the antigen receptors on lymphocytes - have no intrinsic catalytic activity and serve simply as specific recognition units. These receptors use a variety of mechanisms, including adaptor molecules or catalytically active regulatory molecules such asG-proteins (guanosine triphosphatases [GTPases], which hydrolyze GTP) to couple them to their effector signaling elements, which are generally enzymes (often called signaling enzymes or signal transducers), or ion channels (Fig. 25.2). In contrast, other receptors (e.g., the intrinsic tyrosine kinase receptors for insulin and many growth factors; the intrinsic serine kinase receptors for molecules such as transforming growth factor-β) have extracellular ligand-binding domains and cytoplasmic catalytic domains. After receptor ligation, these receptors can directly initiate their signaling cascades by phosphorylating and modulating the activities of target signal-transducing molecules (downstream signaling enzymes). These in turn propagate the growth factor signal by modulating the activity of further specific signal transducers or transcription factors, leading to gene induction (Chapter 23). Furthermore, sensory systems such as vision (Chapter 39), taste, and smell use similar mechanisms of cell-surface membrane receptor–coupled signal transduction (Table 25.1).

4.1 Mineralocorticoid Receptor

Membrane receptors have been identified for several of the steroid hormones. For many years there has been healthy debate on whether the non-genomic actions of aldosterone were mediated by an MR at the plasma rather than the intracellular receptor since these actions were not prevented by MR antagonists spironolactone or canrenone and not mimicked by cortisol (Gekle, Silbernagl, & Wunsch, 1998; Grossmann et al., 2010; Le Moellic et al., 2004; Wehling et al., 1991; Wildling, Hinterdorfer, Kusche-Vihrog, Treffner, & Oberleithner, 2009). While there have been intense attempts to isolate and characterize the membrane receptor for aldosterone, the identity eluded characterization until recently. This was possibly due to early studies using bovine serum albumin (BSA)-coupled aldosterone (Gekle et al., 1998; Le Moellic et al., 2004) which did not consider that BSA can dissociate from aldosterone. In separate studies, atomic force microscopy technology (Wildling et al., 2009) or fluorescence resonance energy transfer (Grossmann et al., 2010) was used; however, the specific location in the plasma membrane could not be identified. We and others have shown that for heart (Mihailidou et al., 1998) and vascular smooth muscle cells (Alzamora, Michea, & Marusic, 2000), the rapid actions of aldosterone were prevented by the MR antagonists suggesting involvement of the intracellular MR.

In further studies (Ashton et al., 2015) we succeeded in separating the non-genomic from genomic signaling by using an aldosterone analog, aldosterone-3-carboxymethoxylamine-TFP ester conjugated to methoxypegylated amine (Aldo-PEG) (Fig. 4). While having similar structure to aldosterone, Aldo-PEG has a larger molecular size and the hydrophilic nature of pegylated amine results in preventing membrane permeability which we confirmed in fractionated H9c2 cells (Fig. 5). In the absence of aldosterone, MR is localized predominantly in the cytoplasm rather than nucleus. While addition of aldosterone changed this distribution from cytoplasm to nuclear MR, whereas the Aldo-PEG did not change subcellular localization, confirming that it cannot enter the cell.

Platelets

Membrane receptors for collagen and other subendothelial and extravascular matrix proteins are present on the platelet membrane and mediate binding of unactivated platelets at sites of injury.3-5 Platelet binding is also mediated by von Willebrand factor (vWF) bridging between collagen and the platelet receptor glycoprotein (GP) Ib. These receptor binding events also transmit an activation signal to the platelets. Full platelet activation also requires stimulation by thrombin, however, which is produced as the coagulation reactions are initiated. The platelet surface receptor for fibrinogen, GPIIb/IIIa, rapidly changes conformation from an inactive to an active form on platelet activation.6 This change in conformation allows platelet aggregates to be stabilized by binding to fibrinogen even before conversion to fibrin begins. Platelet activation also initiates the synthesis of prostaglandins and thromboxanes—compounds that modulate platelet activation and promote vasoconstriction.7

Platelet adhesion and activation at a site of injury, in concert with local vasoconstriction, provides initial hemostasis for small caliber vessels. When hemostasis is achieved by these mechanisms, the subsequent stabilization of the platelet plug in a fibrin meshwork can proceed more effectively than if bleeding continues. Initial hemostasis may be established even if a deficiency of plasma coagulation proteins is present. The platelet plug is insufficient, however, to provide long-term hemostasis, and delayed rebleeding occurs if it is not reinforced by a stable fibrin clot during secondary hemostasis.

Platelets.

Membrane receptors for collagen (glycoprotein [GP] VI) and other subendothelial and extravascular matrix proteins are present on the platelet membrane and mediate binding of unactivated platelets at sites of injury.3–5 Platelet binding is also mediated by von Willebrand factor (vWF) bridging between collagen and the platelet receptor GP Ib. These receptor-binding events also transmit an activation signal to the platelets. Full platelet activation also requires stimulation by thrombin that is produced as the coagulation reactions are initiated. The platelet surface receptor for fibrinogen, GPIIb/IIIa, rapidly changes conformation from an inactive to an active form on platelet activation.6 This conformational change allows platelet aggregates to be stabilized by binding to fibrinogen even before conversion to fibrin begins.

Platelet activation also initiates the synthesis of prostaglandins and thromboxanes—compounds that modulate platelet activation and promote vasoconstriction.7 Platelet adhesion and activation at a site of injury, in concert with local vasoconstriction, provides initial hemostasis for small-caliber vessels. Once hemostasis is achieved by these mechanisms, the subsequent stabilization of the platelet plug in a fibrin meshwork can proceed more effectively than if bleeding continues. Initial hemostasis may be established even if a deficiency of plasma coagulation proteins is present. The platelet plug is insufficient, however, to provide long-term hemostasis and delayed rebleeding occurs if it is not reinforced by a stable fibrin clot during secondary hemostasis. Even after overt bleeding (loss of red blood cells) is stopped by the stable fibrin clot, leakage of plasma proteins from the microvasculature continues. A hemostatic clot structure with a densely packed core of platelets is required to form a tight vascular seal that minimizes the leakage of plasma proteins at a site of injury.8

It is becoming clear that, in addition to providing primary hemostasis following an overt injury, platelets also play more complex and subtle roles in maintaining vascular integrity. It has long been known that platelets maintain endothelial integrity in the microvasculature.9 A failure of this function is responsible for petechiae resulting from thrombocytopenia. However, platelets also directly prevent microvascular bleeding at sites of inflammation10 and angiogenesis11 by mechanisms that are independent of fibrin generation.12

Membrane Receptors

Membrane receptors can be divided into several major groups (Fig. 1-7): (1) seven transmembrane domain G protein–coupled receptors (GPCRs), (2) tyrosine kinase receptors, (3) cytokine receptors, and (4) transforming growth factor-beta (TGF-β) family serine kinase receptors. There are several hundred GPCRs.25 They bind a broad array of hormones, including large proteins (e.g., TSH, PTH), small peptides (e.g., TRH, somatostatin), catecholamines (epinephrine, dopamine), and even minerals (e.g., calcium). These receptors possess seven transmembrane-spanning regions composed of hydrophobic α-helical domains that are connected by extracellular and intracellular loops. After the receptor binds a hormone, these transmembrane domains undergo conformational changes that alter interactions with intracellular G proteins. The G proteins provide a link to intracellular signaling pathways such as adenyl cyclase, phospholipase C, mitogen-activated protein kinases (MAPK), and others. G proteins form a heterotrimeric complex that is composed of various Gα and Gβ-γ subunits. The α subunit contains the guanine nucleotide–binding site and hydrolyzes GTP to GDP. Gα is active when GTP is bound and inactive after hydrolysis to GDP. The β-γ subunits modulate activity of the α subunit and mediate their own effector-signaling pathways. A variety of endocrinopathies result from G protein mutations or from mutations in receptors that modify their interactions with G proteins. For example, McCune-Albright syndrome is caused by somatic mutations in Gα that prevent GTP hydrolysis, thereby causing constitutive activation of the Gα-signaling pathway. Selected mutations in the transmembrane domains of GPCRs can mimic hormone-induced conformational changes, leading to activation of G proteins independent of hormone binding. This type of mutation in the TSH receptor accounts for a significant fraction of solitary autonomously functioning thyroid nodules. Activating mutations in the LH receptor cause LH-independent precocious puberty in boys.

Tyrosine kinase receptors transmit signals for insulin and a variety of growth factors, such as IGF-1, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factors. Ligand binding induces autophosphorylation, leading to interactions with intracellular adaptor proteins such as Shc and insulin receptor substrates (IRS). Depending on the receptor and adaptor complexes, one or more kinases, including the Raf-Ras-MAPK and the Akt/protein kinase B pathways, are activated. The tyrosine kinase receptors play a prominent role in cell growth and differentiation, as well as in intermediary metabolism.

The GH and PRL receptors belong to the cytokine receptor family. Ligand binding induces receptor interactions with intracellular kinases such as the Janus kinases (JAKs), which phosphorylate members of the signal transduction and activators of transcription (STAT) family, and other signaling pathways (Ras, PI3-K, MAPK). The activated STAT proteins translocate to the nucleus and stimulate expression of target genes.

The TGF-β receptor family mediates the actions of TGF-βs, activins, Müllerian inhibiting substance (MIS; also known as anti-Müllerian hormone), and bone morphogenic proteins. This receptor family consists of type I and II subunits, which undergo autophosphorylation after ligand binding. The phosphorylated receptors bind intracellular Smads (named for a fusion of terms for Caenorhabditis elegans sma + mammalian mad). Like the STAT proteins, the Smads serve a dual role of transducing the receptor signal and acting as transcription factors.

3.1 Detection of FSHR self-association at the plasma membrane by cell surface fluorescence resonance energy transfer

Membrane receptor oligomerization has to date been primarily studied using FRET which occurs over intermolecular distances between 1.0 and 10.0 nm and is thus a way to assess protein–protein interaction even under conditions of very small clusters of proteins in the PM (Kenworthy, 2001; Kenworthy & Edidin, 1998; Siegel et al., 2000). The FSHR poses a special problem because, unlike other GPCRs or even the LHCGR which has been studied extensively with FRET assays (Lei et al., 2007), the FSHR is clipped at the C-tail and, therefore, it is not possible to employ fluorescent protein fusions at the carboxyl-terminus of the receptor, as is the standard of practice with other GPCRs. In an alternative approach, it has been possible to employ the mAb 106.105 conjugated to Alexa 568 and to Alexa 647 to successfully detect the presence of FSHR oligomers at the PM (Thomas et al., 2007). The use of differentially labeled mAb 106.105 or mAb 106.105-Fab′ fragments for hFSHR FRET obviates the need for C-tail fusion proteins, which may be useful to detect FSHR–FSHR interactions that occur within the cell but not at the PM level as the C-tail-terminal end of the FSHR (but not that of the LHCGR) is subjected to proteolytic processing during biosynthesis (Thomas et al., 2007). In fact, C-tail epitope tags in mature, cell surface-expressed FSHR usually are undetectable.

The Alexa 568 and Alexa 647 chromofluor pair meets the requisite characteristic to produce FRET, in which the emission spectrum of the donor (Alexa 568) overlaps the absorption spectrum of the acceptor (Alexa 647). The efficiency of energy transfer (E) from donor to acceptor is highly dependent on the distance between the chromofluors, and is often reported as E%. The easiest way to quantify the absolute efficiency of transfer is to measure donor emission before and after photobleaching of the acceptor. The increase in emission (dequenching) of the donor is a direct measure of the FRET efficiency (E), and is calculated from: E% = [1 – (donor emission prior to acceptor photobleach)/(donor emission after acceptor photobleach)] × 100. Analyses are performed directly on captured confocal images, using a cell or its subregion as its own internal standard after photobleaching (Siegel et al., 2000). This procedure is called acceptor photobleaching FRET. The acceptor photobleaching cell surface fluorescence resonance energy transfer (CSFRET) method employed to detect dimerization of the hFSHR is the following:

Human embryonic kidney-293 cells stably expressing the hFSHR and plated in 35-mm Petri dishes with glass coverslip bottoms (MatTek Corp, Ashland, MA) are placed on ice following exchange of tissue culture medium with ice-cold PBS. Cells are then incubated with a 1:1 (μg/μg) mixture of mAb 106.105-Alexa 568 or mAb 106.105-Alexa 647 (directly labeled according to the manufacturer's instructions with Alexa 568 or Alexa 647; Molecular Probes, Inc, Eugene, OR), for 30 min at 4 °C. The cells are washed twice with ice-cold PBS for 5 min, and then fixed with 4% formaldehyde in PBS freshly prepared from a 16% formaldehyde solution. Following fixation, the cells are washed in PBS at RT two times for 5 min. The cells are then covered with PBS and imaged on a Zeiss LSM 510 META confocal microscope system on an Axiomat 200 M inverted microscope equipped with a 63 × 1.4 NA, oil immersion differential interference contrast lens. Alexa 568 images are collected using the 540-nm laser line from a HeNe laser and 545 dichroic mirror and a band pass filter of 565–615 nm. Alexa 647 images are collected using the 633-nm laser line from a HeNe laser and 545 dichroic mirror and a band pass filter of 650–710 nm. The pinhole is set at 1.32 Airy units and a Z resolution of ∼ 2.0 μm. Images may be collected at 12-bit intensity resolution over 512 × 512 pixels at a pixel dwell time of 6.4 μs. FRET is recorded by examining the dequenching of the Alexa 568 in a region of interest (ROI) following photobleaching of the Alexa 647 by the 633 nm HeNe laser line for 30–90 s at maximum power. This irradiation results in greater than 95% photodestruction of the acceptor fluor.

Images may be analyzed using, for example, a LSM FRET tool that is integrated with the LSM 510 collection software (Zeiss, Inc. Thornwood, NY). FRET analyses are performed on ROI drawn directly on captured confocal images. The analysis uses a cell or its subregion as its own internal standard after photobleaching (Siegel et al., 2000).